The Effect Of PH, Temperature On The Green Synthesis And Biochemical .
Husam M. kredy et al /J. Pharm. Sci. & Res. Vol. 10(8), 2018, 2022-2026The effect of pH, Temperature on the green synthesis andbiochemical activities of silver nanoparticles from Lawsoniainermis extractHusam M. kredyDepartment of chemistry, College of Science, University of Dhi-QarAbstractNanotechnology is an innovative technique which includes the design, characterization, production, and application of structures, devices,and systems by controlling shape and size at the nanometer scale. It covers the size range of 1 nm to 100 nm. Silver nanoparticles exhibit newor improved properties depending upon their size, morphology, and distribution. In this study, green synthesis of silver nanoparticles(AgNPs) from silver nitrate (AgNO3) conducted using lawsonia inermis leaves extract as reducing agent in pH and temperature different.The biosynthesized nanoparticles were characterized by UV-Vis at range 300 -800, the results shown an increase in the rate of formation ofsilver nanoparticles with increasing temperature, and there was monodispersive silver nanoparticles were obtained at pH equal to9.Antioxidant activity of silver nanoparticles was tested by (DPPH) and was compared with standard ascorbic acid. The antioxidant test ofthe DPPH showed that these AgNPs could scavenge free radicals at different levels and a high-test inhibition percentage better than that ofascorbic acid , where found to be 72.57% at pH 9, 65.34% at pH 7 for AgNPs, methanolic extract showed antioxidant activity 59.67% ascompared to standard ascorbic acid 62.67% , at a concentration of 100 μg/mL.Key words: Lawsonia inermis, Silver nanoparticles, Antioxidant activity, Antibacterial activity.1. INTRODUCTIONNanotechnology is an innovative technique which includes thedesign, characterization, production, and application ofstructures, devices, and systems by controlling shape and size atthe nanometer scale. It covers the size range of 1 nm to 100 nm[1]. Nanotechnology is nowadays an active domain for thesynthesis of nanoparticles by using natural sources such as plantleaf, stem, bark and root for novel metals such as platinum, gold,and silver and is widely applied in biomedical applications suchas diagnostic, imaging, drug delivery and therapeutics usingmetal nanoparticles [2]. “Nano” is derived from the Greek word“nanos”, meaning “dwarf, tiny, or very small” [3]. Nanoparticlesare the materials with the overall size of 100 nm. Nanoparticlescan be classified as polymeric (natural and synthetic), lipoidal(biodegradable), and metal nanoparticles (iron oxide, goldnanoparticles, and silver nanoparticles) [4]. Nanoparticles ofnoble metals have attracted immense interest incurringapplications in catalysis, electronics, optics, environmental andbiomedical applications due to their quantum confinementeffects, antimicrobial activity and their large reactive surfaces[5].Silver nanoparticles exhibit new or improved propertiesdepending upon their size, morphology, and distribution. Variousapproaches using plant extract have been used for the synthesisof metal nanoparticles [6].Nanoparticle synthesis methods can be classified as bottom-upand top-down. Chemical methods involve the reduction ofchemicals [7], electrochemical procedures [8], and reduction ofphotochemicals. Plant-based synthesis of nanoparticles is incontrast faster, safer and lighter; works at low temperatures; andrequires only modest and environmentally safe components [9].Green synthesis of nanoparticles using plant extracts has severaladvantages over other environmentally green synthesis methods,because plants are broadly distributed, readily scalable, easilyavailable, safe to handle and less expensive [10]. In this research,the methanolic extract of lawsonia inermis was used for rapid,simple and biosynthetic synthesis of silver nanoparticles.Lawsonia inermis (Henna) is a dwarf shrub belongs toLythraceae family. L. inermis is generally used as traditionalmedicine worldwide to treat various diseases such as edema,bronchitis, rheumatism, small pox, spermatorrhoea, menstrualdisorders and hemorrhoids [11]. L. inermis leaves consist of adiverse range of biomolecules, which formulate it as a richsource of different types of medicine [12]. Improvements inchemical composition, size, shape, and dispersity ofnanoparticles would permit the use of nanobiotechnology in avariety of other applications [13]. The hydroxyl and carbonylgroups present in carbohydrates, flavonoids, terpenoids, andphenolic compounds are powerful reducing agents that may beresponsible for the bio reduction of Ag ions necessary forAgNP synthesis [14].Several characterization techniques for analysis, certain tabulardata representing source, shape and size and informationregarding various applications. Ag nanoparticles werecharacterized by UV-Vis Spectra and SEM techniques.Nowadays, the most important applications of silvernanoparticles in biotechnology science correspond to theirantibacterial and antioxidant activities [15]. The antimicrobialactivity of AgNPs is widely recognized, though their activity canchange with physical characteristics of the nanoparticle, such asits shape, mass, size, and composition, and conditions of itssynthesis, such as by pH, ions, and macromolecules. Theirshapes can be relevant to their antibacterial activity [16]. In thepast decades, nanotechnology has been used to prepareantioxidant products using minerals including silver, gold,cerium oxide and platinum [17].The study aimed to synthesize silver nanoparticles usingmethanolic extract of L. inermis (2ml of AgNO3: 0.4ml ofextract). Moreover, to Characterize the prepared AgNPs usingSEM, UV-Vis spectroscopy. Furthermore, studying andevaluating the antibacterial and antioxidant activity of silvernanoparticles.2. EXPERIMENTAL2.1 Collection of L. inermis LeavesLawsonia inermis leaves were collected from North West of thecity of Nasiriyah in southern of Iraq in march, 2017. TheCollected leaves were washed and dried in the shade at roomtemperature for 10 days, then crushed to obtain powder. Thesubstance was kept in the fridge at 4 C until use.2.2 Preparation of L. inermis Leaves ExtractFive gram of l. inermis leaves powder was weighed andmixed in 100 ml of methanol and the mixture was heatedfor 10 minutes then filtered through Whattman filter paperNo.1.The filtrate was collected and stored at 4 C for furtheruse.2022
Husam M. kredy et al /J. Pharm. Sci. & Res. Vol. 10(8), 2018, 2022-20262.4 UV-Visible spectrophotometerThe optical property of AgNPs and The reduction of pure Ag ion were monitored by measuring the UV-Visible spectrum ofthe reaction mixture. UV-Vis spectral analysis was performed byusing UV-Vis spectrophotometer UV-1700 (Shimadzu, Tokyo,Japan) that was operated in the scanning range of 300-800 nm.2.5 Antibacterial ActivityAntibacterial activity of biologically synthesized AgNPs of L.inermis was determined by agar well diffusion method againstdifferent pathogenic microorganismsEscherichia coli,Pseudomonasaeruginosa,Streptococcus pneumonia,Klebsiella pneumoniae, Staphylococcus aureus and Bacillussubtilis. Muller Hinton Agar plates were used and swabbed withpathogenic organisms from fresh cultures (105-106 CFU/mL)using a sterile cotton swab. The plates were then incubated at 37 C for 24 h. At the end of the incubation period, the zones ofinhibition were measured to the nearest millimeter [18]. Theinhibition zone is the area surrounding the hole with no growthof inoculated microorganisms.2.6 Antioxidant ActivityThe antioxidant activity of AgNPs was determined by DPPHassay method [19]. The capability to scavenge the DPPH radicalwas calculated using the following equation:𝐴𝑐 𝐴𝑠DPPH Scavenged (%) 𝐴𝑐 𝑥 100Where Ac and as are the absorbance of the control and testsample, respectively, after 30 min measuring at 517 nm.3. RESULTS AND DISCUSSION3.1 Visual observationThe color of the L. inermis extract was dark brown before itstreatment with silver nitrate solution, but after the reaction itturned to yellowish brown (Fig. 1), indicating the formation ofAg-NPs due to reduction of silver ions by active moleculespresent in the extract. This color is attributed to surface plasmonresonance, which is a size-dependent property of NPs [20].Silver nitrate AgNO3 ;L. inermis extract ;Silver nanoparticles AgNPsFig 1. Synthesis of silver nanoparticles by Visual observationindicator.3.2 UV-Visible spectrophotometerMonitoring the process of the bio reduction of silver ions toAgNPs was applied in this study by UV-vis spectroscopy. UVvis spectroscopy might be used to detect the size and shapecontrolled NP in aqueous suspensions [21]. UV-Vis spectroscopywas used to follow the reactions process and characterize theoptical properties of produced nanoparticles. The formation andoptimization of Ag NPs was monitored using UV–Visspectroscopy by measuring the absorbance in the range of 300–800 nm, by varying the temperature and pH. UV–Vis absorptionmeasurements in the range 300–800 nm can provide deep insightinto the optical properties of the formed nanosized silverparticles. The change in color indicates the formation of Ag NPswhich was further confirmed by the appearance of the SPR bandbetween 400 to 500 nm [22]. The UV-VIS Spectral analysis ofthe green synthesized nanoparticles was observed a sharp peakaround 386 – 450 nm indicates the formation of silvernanoparticles, which was identified as “surface Plasmonresonance band” and this band is ascribed to excitation ofvalence electrons. The position of absorption band also mainlydepends upon dielectric constant of the medium and surfaceadsorbed species. The shape of the band was symmetrical,suggesting uniform scattering of spherical shape nanoparticles[23]. There are factors affecting the intensity absorption bandand thus affect the synthesis of nanoparticles. This effect isexplained as below:The effect of temperatureThe affect Temperature is one of the factors influencing thesynthesis of silver nanoparticles. This was confirmed by studyingthe UV-Vis spectra shown from fig.2 at three differenttemperatures 25 C, 35 C and 45 C. Where we observe anincrease in the rate of formation of silver nanoparticles withincreasing temperature.Absorbance2.3 Synthesis of silver nanoparticles AgNPsFor synthesis silver nanoparticles, 0.4 ml of l. inermis leavesextract was added to 2ml of 1 mM AgNO3 solution in test tubewith vigorous shaking for the bio reduction of Ag ions. Thecolor change indicated confirmation for the formation of silvernanoparticles.Optimization factorsThe Effect of pHpH of the reaction mixture was maintained at 4, 7 and 9,respectively, by using (0.1 N) Hcl and (0.1 N) NaOH. Theabsorbance of the resulting solutions was measuredspectrophotometrically.The Effect of temperatureTo study the effect of temperature on the synthesis of AgNPs, atypical sample was synthesized at 25 C, 35 C and 45 C.Electronic absorption spectra of the aqueous colloidalsuspensions were recorded at each temperature range.10.90.80.70.60.50.40.30.20.1045 C35 C25 C200400600Wave length (nm) rFig. 2: The effect of temperature on wavelength and absorbance forAgNPsUV spectra indicated that the wavelength was higher at lowertemperature, but the wavelength shifted to a lower value athigher temperature resulting in the formation of smaller silvernanoparticles at higher temperature, whereas at higherwavelength the size of silver nanoparticles increased. Themaximum absorbance was observed at 470, 452 and 431 nm at25, 35 and 45 ᴼC, respectively. It means at higher temperature,reactants are consumed rapidly, resulting in the formation ofsmaller nanoparticles [24].The effect of pHThe effect of pH was studied in three different conditionsincluding acidic, neutral and basic forms. The fig 3 shows theeffect of changes in pH on UV-Vis spectra of silver nanoparticlessynthesized in pH 9.0, 7.0 and 4.0, respectively. The highest2023
Husam M. kredy et al /J. Pharm. Sci. & Res. Vol. 10(8), 2018, 2022-2026color intensity of the reaction mixture was observed at pH9.there was no reaction at a pH value equal to 3, butmonodispersive silver nanoparticles were obtained at pH equal to9[25].pH 910.90.80.70.60.50.40.30.20.10pH 7AbsorbancePH 4200400600Wave length (nm) rFig.3: The effect of pH on wavelength and absorbance for AgNPs3.3 DPPH radical-scavenging activityIn the present study, In order to determine the extent ofscavenging effect, antioxidant activity of L. inermis extract andAgNPs were comparatively studied by DPPH method. Theresults were expressed as % RSA (radical scavenging activity)[26]. The DPPH reducing ability of the AgNPs was assessed byobserving color change and the control does not show any colorchange , which produced violet color in methanol solution.it wasreduced to yellow colored product, diphenyl picrylhaydrazine[27]. DPPH scavenging assay exhibited effective inhibitionactivity of AgNPs at pH 9 when compared with AgNPs atpH 7, extract and the standard Ascorbic acid. The antioxidantproperty of L. inermis lawsonia leaves extract was found to beless effective. Our silver particles have shown much betteractivity. AgNPs synthesized using L. inermis leaves extract havethe highest recorded radical scavenging activity of .72.57% atpH 9, 65.34% at pH 7, methanolic extract showed antioxidantactivity 59.67% as compared to standard ascorbic acid 62.67%,at a concentration of 100 μg/mL. As showing in fig.4Antioxidant Activity100Ascorbic acid90AgNPs (s2)80% DPPH InhibitionLawsonia inermis extractAgNPs (s1)70603.4 Antibacterial ActivityThe potential antibacterial activity of biologically synthesizedAgNPs was detected and compared with the antibacterial activityof antibiotic (ciprofloxacin) because it is a strong antibiotic andshowed good antibacterial activity against tested bacteria.However, the antibacterial activity was species-dependent.DMSO solvent was used because it showed no efficacy on allthe bacteria strain used. The susceptibility of bacteria to plantextracts varied according to bacterial strains and species, whichwas well documented [28].The disc diffusion method, a most commonly used technique toaccess the antimicrobial activity, has been employed by manyresearchers to confirm antibacterial action of the AgNPssolution. The results of AgNPs showed higher ability to suppressthe microbial growth than methanolic extract. The maximuminhibition of AgNPs was recorded against S. aureus (22) mm,(20) mm for E.coli, (21) for P. aeruginosa, (17) mm for K.pneumoniaeand (21) of B. subtilis for AgNPs, and incomparison with antibiotic (ciprofloxacin) showing synergisticeffect, the Inhibition zones of ciprofloxacin antibiotic discs were0,0,30,30,28,22 mm for P. aeruginosa, E.coli, B. subtilis, S.aureus, K. pneumoniae and S. pneumoniae respectively asshowed in table 1.Table1. The inhibitory ability of silver nanoparticles against bacteriastrains. (Zone of inhibition)Name occusGram19220pneumoniaepositiveGramBacillus subtilis21300positiveGenerally, biologically synthesized AgNPs showed goodantibacterial capability against Gram-positive than Gramnegative bacteria. The difference in the effect of the antibacterialis due to the difference in the structure of the cell wall, where thecell wall of the gram positive bacteria contains a single layer,while the cell wall of Gram-positive bacteria composed of a rigidthicker multiple layer of peptidoglycan, as it prevented thenanoparticles from entering into cell wall. The difference in thestructure of the cell wall is due to its cell wall containingcompounds such as lipopolysaccharides, lipoprotein and proteinlipid for gram-negative bacteria while Gram-positive bacteriawall, have a lipid content that makes them more permeable andtherefore more effectively to gram-negative bacteria [29].504030201012345concentration μg/mlFig.4 The free radical scavenging activity of AgNPs at differentconcentration when compared to the standard ascorbic acid and L.inermis extract.Fig.5 Antibacterial activity of AgNPs against S. pneumoniae.2024
Husam M. kredy et al /J. Pharm. Sci. & Res. Vol. 10(8), 2018, 2022-2026Fig. 6 Antibacterial activity of AgNPs against S . aureus.Fig. 10 Antibacterial activity of AgNPs against K. pneumoniae.CONCLUSION:The antibacterial activity was examined against six types ofstrain bacteria, Gram positive bacteria (S. aureus, B. subtilis andS. pneumoniae) and Gram negative bacteria (E.coli ,P.aeruginosa and K. pneumoniae ) by using the agar disk diffusionmethod. The results showed that silver nanoparticles had a strongeffect against Gram positive bacteria and it is more than Gramnegative bacteria.REFERENCES1.2.Fig 7. Antibacterial activity of AgNPs against E.coli.3.4.5.6.7.8.Fig.8 Antibacterial activity of AgNPs against P. aeruginosa.9.10.11.12.13.14.Fig.9 Antibacterial activity of AgNPs against B. subtilis.15.Raj, S.; Jose, S. and Sumod, U.(2012),Sabitha M. Nanotechnologyin cosmetics: opportunities and challenges. Journal of Pharmacyand Bioallied Sciences. 4(3):186–193.Siavash, I .(2011), Green synthesis of metal nanoparticles usingplants, Green Chem, 13(10):2638–2650.Rai, M.; Yadav, A. and Gade, A. (2008), Current trends inphytosynthesis of metal nanoparticles. Critical Reviews inBiotechnology, 28(4) , 277-284.Kumar, D.; Jain, N.; Gulati, N. and Nagaich, U.(2013),Nanoparticles laden in situ gelling system for ocular drug targeting.Journal of Advanced Pharmaceutical Technology andResearch.4(1):9–17.Sharma, V.; Yngard, R. and Lin, Y.(2009), Silver nanoparticles:green synthesis and their antimicrobial activities. Adv ColloidInterface Sci. 145(1-2):83-96.Jha, A. and Prasad, K. (2010) Green synthesis of silvernanoparticles using Cycas leaf. Inter. J .Green Nanotechno: Phys.Chem 1:110 – 117.Guzmán, M.; Dille, J. and Godet, S. (2009) Synthesis of silvernanoparticles by chemical reduction method and their antibacterialactivity. Int. J. Chem. Biomol. Eng .2(3):104–111Rodríguez-Sánchez, M.; Blanco, M. and López-Quintela, M. (2000)Electrochemical synthesis of silver nanoparticles. J. Phys. Chem.B., 104(41):9683 – 9688.Goodsell ,D. (2004), Bionanotechnology: lessons from nature,Wiley-Liss, Hoboken David S .Mittal, A.; Chisti, Y. and Banerjee, U.(2013), Synthesis of metallicnanoparticles using plant extracts. Biotechnol. Adv, 31(2):346–356.Chaudhary, G.; Goyal, S. and Poonia, P.(2010), Lawsonia inermisLinnaeus: a phytopharmacological review, Int. J. Pharm. Sci. DrugRes. 2(2):91–98.Hsouna, A.; Trigui, M.; Culioli, G.; Blache, Y. and Jaoua, S.(2011),Antioxidant constituents from Lawsonia inermis leaves: isolation,structure elucidation and antioxidative capacity, Food Chem.125(1): 193–200.Bai, H.; Zhang, Z.; Guo, Y. and Yang, G.(2009), Biosynthesis ofcadmium sulfide nanoparticles by photosynthetic bacteriaRhodopseudomonas palustris. Colloids Surf. B Biointerf 70(1):142– 146Ajitha, B.; Reddy, Y. and Reddy, P. (2015) , Green synthesis andcharacterization of silver nanoparticles using Lantana camara leafextract. Mater. Sci. Eng. C 49: 373 – 381Allafchian, A.; Jalali, S.; Bahramian, H. and Ahmadvand, H.(2016),Preparation, characterization, and antibacterial activity of NiFe 2 O2025
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The Effect of pH . pH of the reaction mixture was maintained at 4, 7 and 9, respectively, by using ) Hcl and (0.1 N) (0.1 NNaOH. The absorbance of the resulting solutions was measured spectrophotometrically. The Effect of temperature . To study the effect of temperature on the synthesis of AgNPs, a
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4.3.1 Effect of Temperature at pH 4.5 57 4.3.2 Effect of Temperature at pH 5.0 58 4.3.3 Effect of Temperature at pH 5.5 59 4.3.4 Effect of Temperature at pH 6.0 60 4.3.5 Effect of Temperature at pH 6.5 61 4.3.6 Combination Effect of Temperature on Enzymatic Hydrolysis 62
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